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PubMed1976

[Quantitative structure comparison of protein surfaces by micro-complement fixation].

Microcomplement fication (MCF) is a rapid and most sensitive method for the quantitation of slight modifications on protein surfaces not accessible by other immunologic means. Single amino acid substitutions (0.7 percent sequence differences) are readily detected. A firm relationship between sequence information and immunologic distance as measured by MCF could be established. The rate of molecular evolution of homology proteins can be derived from MCF measurements without prior knowledge of primary structures.

Amino Acids↗
PubMed1984

Rapid, homogeneous phase, liposome-based assays for total complement activity.

A simple, rapid assay for determining total complement activity has been developed. Complement activity is quantitated spectrophotometrically by measuring the amount of liposome-encapsulated enzyme unmasked by the action of anti-Dnp antibody and complement on Dnp-tagged liposomes. The assay is homogeneous in nature and is nonisotopic. The activity of complement in guinea pig serum has been measured and shown to be proportional to complement concentration. The assay was modified to measure the complement-fixing titer of anti-Dnp antibody preparations. We have compared two monoclonal anti-Dnp antibodies (IgG1 and IgM) for their ability to fix complement. The IgM antibody preparation was 450-fold more effective than the IgG1 preparation in mediating complement-dependent damage to Dnp liposomes. In addition, the test was modified to measure complement fixation by soluble antigen-antibody complexes. This complement fixation format is capable of detecting 2 pmol Dnp antigen.

2,4-Dinitrophenol↗
PubMed1991

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals.

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.

Antibodies, Monoclonal↗
PubMed1996

Phospholipids in meibomian gland secretion.

The bulk of the lipid layer overlying the aqueous portion of the precorneal tear film is composed of polar and nonpolar components. The nonpolar lipids have been the subject of numerous studies; however, the polar lipids have remained relatively uncharacterized. The polar lipids are thought to contain surfactant phospholipids that are critical to the spreading of a lipid film over the aqueous layer, by providing an interface between this layer and the nonpolar lipids. The purpose of the present study is to identify and quantitate the phospholipid complement of meibomian gland secretion which provides the tear film with phospholipids. Meibomian gland secretion was collected from rabbits and phospholipids identified and quantitated by 31P nuclear magnetic resonance spectroscopy. Ten phospholipids were detected from meibomian gland secretion: diphosphatidylglycerol, dihydrosphingomyelin, ethanolamine plasmalogen, phosphatidylethanolamine (PE), phosphatidylserine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol, alkylacylphosphatidylcholine, and phosphatidylcholine (PC). The two major phospholipids were PC and PE, together comprising nearly 60% of the total phospholipid profile. The nature and relative concentrations of the meibomian gland secretion phospholipids are congruous with a surfactant role at the aqueous-lipid interface and, considering the physical chemistry of the tear film, suggest that the phospholipids should be organized in a very flat or planar configuration.

Animals↗
PubMed1977

Complement fixing site of human IgG1.

A number of chemical modifications were made to purified human IgG1. The effects of these modifications on the complement activity of the immunoglobulin were studied using aggregation on latex and measuring the consumption of the complement by CH50-quantitation. Tryptophan and tyrosine are implicated in the complement fixing site of this human immunoglobulin, and an arginine moiety probably provides a binding site for the complement.

Adsorption↗
PubMed1984

Protein loss induced by complement activation during peritoneal dialysis.

A variable loss of macromolecules during peritoneal dialysis has been noted in both humans and experimental animals. We investigated the potential role of the complement system for inducing protein loss during peritoneal dialysis, both to shed light on clinical variability of protein loss and to develop a model for quantitatively studying complement-induced microvascular protein leakage. Rats received intra-arterial injections of a fluorescent dye conjugated to rat serum albumin and underwent a 3.5-hr series of 15-min peritoneal dialysis exchanges. After the control exchanges, rats received either intra-arterial zymosan-activated rat serum, saline, unactivated rat serum, or endotoxin; other rats received an intraperitoneal injection of endotoxin, histamine, phenylephrine, or nitroprusside. The drainage volume from each exchange was measured, and the concentrations of labeled albumin, total protein, and urea were determined by spectroscopy. Zymosan-activated rat serum and endotoxin injections (both intraperitoneal and intra-arterial), each of which may activate the alternative pathway of complement, produced a dramatic increase in dialysate protein concentrations. In addition, histamine, which is a vasodilator but which may also be involved as a mediator of the activated complement system and/or endotoxemia, also produced an increase in dialysate protein concentrations. On the other hand, drugs which may alter peritoneal blood flow such as the vasodilator nitroprusside or the vasoconstrictor phenylephrine, did not affect dialysate protein concentrations. These data suggest that activation of the alternative pathway of complement may cause variation in protein loss during peritoneal dialysis and that in some situations, pharmacological control of this system could be an important therapeutic consideration.

Animals↗
PubMed1981

Rocket immunoelectrophoresis of C4 and C4d. A simple sensitive method for detecting complement activation in plasma.

Activation of complement component C4 has recently been measured by the quantitation of C4 and C4d (a cleavage fragment of C4) by electroimmunodiffusion in gels containing specific precipitating antibodies for C4 and C4d (Rocket immunoelectrophoresis, RIE). Quantitative measurements of the complement component C4 and its fragment C4d were determined in rocket immunoelectrophoresis (RIE) and compared with measurements of total hemolytic complement activity (CH50) or concentrations of C4 as determined by single radial immunodiffusion (RID). This newly developed RIE assay shows activation of the classical complement pathway and involves electroimmunodiffusion in gels containing specific precipitating antibodies for C4 and its cleavage fragment, C4d. In 37 plasma samples, excellent correlation was demonstrated between the C4 in RIE and CH50 (r = .70) and C4 by RID (r = .87). In vivo activation of C4 was determined by measuring the ratio of C4d to C4; 21 of the plasma samples assayed had ratios greater than 1.1 indicating activation of C4. In 13 of the plasma samples there were correspondingly low CH50 values, whereas 8 had normal CH50 levels. Therefore, activation can be detected in those instances when other measurements (CH50 and C4 quantitation) are normal. Thus the RIE assay for plasma C4 activation appears to be the most sensitive method available for assessing in vivo activation of the classical pathway of complement.

Animals↗
PubMed1980

Hemolytic complement in nonhuman primates.

Hemolytic serum complement activity was quantitatively compared in baboons, squirrel monkeys, cebus monkeys, and cotton-top marmosets. Squirrel monkeys showed the highest activity, and marmosets had the lowest activity. The complement level in squirrel monkeys and tenfold greater than marmosets and almost four times higher than that of man. Cebus monkeys had levels most similar to that of man while the baboon exhibited activity almost as low as that of the marmoset.

Animals↗
PubMed1983

A quantitative microassay for leukocyte chemotaxis, using a microscopic slide system with complement-activating yeast particles as gradient source.

A simple quantitative microassay was developed for studying polymorphonuclear leukocyte (PMNL) chemotaxis under conditions where the number of available cells is a limiting factor, e.g., pustules, neutropenia, small children and cerebrospinal fluid. PMNL suspensions are placed on glass slides to which fluorescein-labeled yeast particles have been fixed. After adherence, normal human serum is added to the slides. Owing to complement activation, a chemotactic gradient which attracts the adherent PMNL is formed around the yeast particles. The number of PMNL-associated yeast particles in the presence of normal serum is scored, and compared with cells migrating in the presence of inactivated serum or in the absence of serum. A locomotory index is calculated as the number of yeast particles associated with PMNL divided by the total number of yeast particles.

Blood Physiological Phenomena↗
PubMed1994

Autogenic training: a narrative and quantitative review of clinical outcome.

This review of controlled outcome research on Autogenic Training complements the literature by pooling narrative and quantitative approaches, by including only studies with experimental controls, by integrating the English and German literature, and by adding research findings published since the last review. Whereas previous reviews have already reported positive effects of Autogenic Training for migraine, insomnia, and test anxiety, additional supportive findings for angina pectoris, asthma, childbirth, eczema, hypertension, infertility, Raynaud's disease, and recovery from myocardial infarction are discussed here. The impact of protocol variations on outcome is described, and the specificity of Autogenic Training relative to other stress management techniques is highlighted. Quantitative findings suggested that Autogenic Training was associated with medium-sized pre- to posttreatment effects ranging from d = .43 for biological indices of change to d = .58 for psychological and behavioral indices thus matching effect sizes for other biobehavioral treatment techniques like biofeedback and muscular relaxation. Length of treatment did not affect clinical outcome. The discussion emphasizes how narrative and quantitative strategies complement one another.

Autogenic Training↗
PubMed1983

Humoral defence factors in the breast milk of Ethiopian women with leprosy and healthy controls.

Secretory IgA, lactoferrin, albumin, and total protein were quantitated in colostrum and milk samples obtained from 215 Ethiopian nursing mothers over a period ranging from 1 day to 2 yr postparturition. IgG, IgM, C3, and C4 complement components were quantitated in 11 day 1 samples. The subjects were classified into three groups: lepromatous leprosy, borderline lepromatous leprosy, and a nonlepromatous group consisting of women with tuberculoid leprosy and healthy controls. Results obtained from the above groups were also compared with a group from Edinburgh. No major variation in levels of secretory IgA, lactoferrin, albumin, and total protein was found between the three groups of Ethiopian women. Results from the Edinburgh group were significantly higher, mainly in the level of total protein. When the individual proteins were expressed as a percentage of the total protein, there was no difference between the milk samples from the Ethiopian and Edinburgh mothers.

Albumins↗
PubMed1986

The role of complement in inflammation during experimental pneumococcal meningitis.

The mechanism whereby an effective bactericidal inflammatory reaction develops in the subarachnoid space is not clearly defined. While normal cerebrospinal fluid is deficient in complement, immunoglobulin and leukocytes, these serum components appear in cerebrospinal fluid (CSF) during the course of bacterial meningitis. Using a rabbit model of pneumococcal meningitis we examined the role of the alternate complement pathway in three early events important to the defense of the subarachnoid space: leukocyte chemotaxis, phagocyte mediated bacterial killing, and clearance of bacterial components from the cerebrospinal fluid space. Rabbits treated with cobra venom factor to deplete complement were inoculated intracisternally with encapsulated (type II or XIX) pneumococci. Following complement depletion, there was a dramatic (at least 100-fold) decrease in the LD50 for these strains. Nevertheless, complement depletion did not affect the magnitude of CSF leucocytosis or the rate of clearance of bacterial particles from CSF. A short delay in the appearance of leukocytes in CSF was found in the absence of complement. The major effect of complement depletion, however, was to diminish the efficiency of leukocyte mediated killing of encapsulated bacteria in the CSF. Although the short delay in the onset of leukocytosis in the complement depleted animals is consistent with a chemotactic role of complement in the normal animal, the quantitatively normal leukocytosis in the complement depleted rabbits clearly indicates that important chemotaxins other than complement function in CSF. Inhibition of leukocytosis by indomethacin and diclofenac suggests that metabolite(s) of the arachidonic acid pathway may perform such a chemotactic role. A major role of complement in the defense of the subarachnoid space appears to be as an opsonin needed for the effective bactericidal activity of leukocytes. It is the lack of this function that best explains the greatly decreased LD50 value of encapsulated pneumococci in the complement depleted animal.

Animals↗
PubMed1987

Competitive enzyme-linked immunoassay for the quantitation of platelet-associated immunoglobulins (IgG, IgM, IgA) and complement (C3c, C3d) with polyclonal and monoclonal reagents.

A competitive enzyme-linked immunoassay (CELIA) was developed for the quantitation of platelet-associated immunoglobulins and complement proteins. The use of unlabeled polyclonal rabbit or monoclonal antibodies to human immunoglobulins and enzyme-labeled anti-mouse or anti--rabbit IgG (double-step technique) resulted in a higher sensitivity compared to the single-step technique using only enzyme-labeled anti-human immunoglobulin antibody preparations. Sensitivity and results obtained by both techniques were compared. The range of normal values for platelet-associated IgG, IgM, IgA, C3c and C3d was assessed upon a large number of normal blood donors. When platelet-associated IgG was concomitantly assayed with polyclonal and monoclonal anti-IgG by the double-step technique on platelets obtained from normal donors and thrombocytopenic patients, identical results were obtained with both reagents. Problems related to the quantitation of immunoglobulins on platelets with different assays and antibody preparations are discussed.

Antibodies, Monoclonal↗
PubMed1997

Decreased C4 complement component serum levels correlate with the degree of emphysema in patients with chronic bronchitis.

Patients with COPD who fulfill the diagnostic criteria of chronic bronchitis have been shown to exhibit lower serum levels of complement components C3 and C4 than healthy subjects, and this may indicate sustained complement activation as a result of recurrent respiratory tract infections. Since activation of complement leads to influx of inflammatory cells into the lung parenchyma with subsequent release of elastases and oxidants that cause damage to elastic lung tissue, we postulated that there might be a quantitative relationship between complement consumption and degree of elastic tissue destruction. In this study, we tried to investigate possible correlations between serum levels of C3 and C4 and degree of emphysema among patients with COPD of the bronchitic type. We studied 20 patients with chronic bronchitis aged 68+/-1 years (mean+/-SEM) without significant fluctuations of serum C3 and C4 levels over a 3-month period by performing detailed lung function tests, recording of emphysema score in chest radiogram, and the incidence of infective exacerbations during the past 3 years. Measured C3 and C4 serum levels were 124+/-9 and 28.5+/-2 mg/dL, respectively, lower than the respective levels in control subjects (141+/-3 and 39+/-2 mg/dL, respectively). Significant correlations were observed between levels of C4 and (1) incidence of respiratory tract infections during the past 3 years (r=-0.747, p<0.001), (2) radiologic emphysema score (r=-0.936, p<0.001), and (3) various functional indexes, such as midexpiratory flow rate, percent of predicted (r=0.629, p<0.01), forced expiratory flow rate at 50% of vital capacity, percent of predicted (r=0.606, p<0.01), residual volume/total lung capacity ratio (r=-0.651, p<0.01), and the exponential constant of static pressure-volume curve (r=-0.606, p<0.01). These results suggest that patients with chronic bronchitis with the lowest levels of C4 are those experiencing more frequent respiratory infections, tend to have more signs indicative of emphysema in their chest radiograph, have a more prominent small airways dysfunction and gas trapping, and present a greater defect in lung elastic recoil.

Aged↗
PubMed1987

A prospective study on infection with cytomegalovirus in renal allograft recipients immunosuppressed with cyclosporine A and low dose prednisone.

To investigate the course of infection with cytomegalovirus (CMV) in renal allograft recipients treated with cyclosporine A, 10 patients were followed for 1 year after transplantation. Virus cultures from blood, urine and throat washings were performed employing a quantitative technique. Complement-fixing and IgM antibodies to CMV were measured at scheduled intervals. The incidence 90% and course of CMV infections were found not to differ from those reported in patients receiving conventional immunosuppressive therapy. The quantitative virus cultures showed a consistent pattern with viremia most prominent at the beginning of an infection, and the highest concentration found in the one patient who developed symptoms of viral disease. It is suggested that information about the concentration of virus in a specimen will improve the diagnostic value of virus cultures in this group of patients.

Adolescent↗
PubMed1991

Complement activation and depletion during LDL-apheresis by heparin-induced extracorporeal LDL-precipitation (HELP).

The heparin-induced extracorporeal elimination of low density lipoproteins (LDL) is a well-established clinical procedure to markedly reduce cholesterol levels. The biocompatibility of this artificial filter system (HELP) was investigated by quantitation of representative complement proteins within the extracorporeal circuit using established ELISA procedures, based on monoclonal antibodies recognizing exclusively either native (C6, C7) or activated proteins (act.C3, C5a, TCC). HELP was found to be a self-limiting extracorporeal system with respect to complement activation, since act.C3 and TCC, generated mainly at the plasma filter, were partially adsorbed to the following HELP specific filters to concentrations which were lower than those obtained before the plasma filter. C5a, which increased 14.5-fold at the plasma filter was not eliminated by the following filters; however, elevated levels were not found in the patients at the end of apheresis and no leucocytopenia was observed.

Adult↗
PubMed1979

A rapid and simplified technique for the assay of chicken hemolytic complement.

The technique described is a modification of a qualitative hemolytic radial diffusion technique. The test involves the use of sensitized sheep erythrocytes that have been incorporated into agarose. Tube dilutions were made of chicken serum and samples of each dilution were placed into wells cut in the agarose. The test is quantitative for hemolytic complement in that the highest dilution showing visible hemolysis of sensitized erythrocytes in agarose is determined to be the endpoint for that serum sample. The test as compared with the standard tube assay was determined to be less sensitive by approximately one dilution. The advantages of speed, simplicity, and cost more than offset the decrease in sensitivity of the test.

Animals↗